INTRODUCTION Ryanodine receptors (RYR) are a family of intracellular Ca 2 release channels present in a variety of muscle and non-muscle cells. A combination of molecular biology, biochemical and biophysical approaches have led to the identification of three RYR isoforms: type 1 is mainly expressed in skeletal muscle and restricted areas of the brain; type 2 is found primarily in the heart and cerebel- lum and type 3 has been reported to be present in a vari- ety of cell types (for review see [1]). In skeletal muscle depolarization of the transverse tubules leads to release of calcium from sarcoplasmic reticulum terminal cisternae [2–4]. The key molecular components of this mechanism are two calcium channels, namely the dihydropyridine receptor (DHPR) and the RYR1 which are localized in the transverse tubules and in the sarcoplasmic reticulum junctional face membrane, respectively [5–7]. A direct interaction between the DHPR and the RYR1 is thought to be the molecular basis of the signalling pathway underlying skeletal muscle excitation-contraction [8–11]. Research Functional properties of EGFP-tagged skeletal muscle calcium-release channel (ryanodine receptor) expressed in COS-7 cells: sensitivity to caffeine and 4-chloro-m-cresol S. Treves, 1 P. Pouliquin, 2 L. Moccagatta, 2 F. Zorzato 1,2 1 Departments of Anesthesiology and Research, Kantonsspital Basel, Hebelstrasse 20, Basel, Switzerland 2 Dipartimento di Medicina Sperimentale e Diagnostica, Sezione di Patologia Generale, Università degli Studi di Ferrara, Via Borsari 46, 44100 Ferrara, Italy Summary We constructed and expressed in COS-7 cells, three E-green fluorescent protein (EGFP) tagged recombi- nant skeletal muscle ryanodine receptors (RYR). EGFP was tagged to (i) the NH 2 -terminus ( n EGFP-RYR FL ) and to (ii) the COOH-terminus ( c RYR FL -EGFP) of the full length RYR; we also tagged the EGFP to (iii) the NH 2 -terminus of a truncated version of the RYR ( n EGFP-RYR Bhat ) lacking the bulk of the protein. The fluorescent pattern EGFP with all three constructs colocalize with that of an endoplasmic reticulum (ER) membrane tracker fluorescent dye, indicating that the RYR constructs are targeted to ER membranes. Our results show that: (i) COOH-terminal tagging abolishes the sensitivity of the RYR to caffeine, whereas the presence of EGFP at the NH 2 -terminus does not affect caffeine sensitivity and (ii) 4-Cl-m-cresol sensitivity is lost both with the trun- cated n EGFP-RYR Bhat and the n EGFP-RYR FL , while COOH-terminal tagging does not affect sensitivity to 4-chloro-m-cresol. The dose–response curves of caffeine-induced calcium release of n EGFP-RYR FL differ from those of the truncated n EGFP-RYR Bhat . Maximal calcium release was approached at 10 mM caffeine with the n EGFP-RYR FL , while cells expressing the n EGFP-RYR Bhat construct displayed a bell shaped curve and the maximal concentration for caffeine- induced calcium release was 5 mM. Equilibrium [ 3 H]-ryanodine binding confirmed the calcium photometry data. Our results demonstrate that EGFP tagging modifies the pharmacological properties of RYR, and suggest that 4-chloro-m- cresol and caffeine act through different mechanisms and probably interact with different sites on the RYR calcium release channel. © 2002 Elsevier Science Ltd. All rights reserved 1 Cell Calcium 2002 31(1), 1–12 © 2002 Elsevier Science Ltd. All rights reserved doi: 10.1054/ceca.2001.0252, available online at http://www.idealibrary.com on Received 3 July 2001 Revised 27 September 2001 Accepted 27 September 2001 Correspondence to: Susan Treves, Departments of Anesthesia and Research Lab 408, ZLF Kantonsspital Basel, Hebelstrasse 20, Basel 4031, Switzerland. Tel.: 41-61-265-2373; fax: 41-61-265-3702; e-mail: susan.treves@unibas.ch